1. Field of the Invention
The present invention relates to a method for extracting IgY (γ-livetin) from egg yolk and, more particularly, to an easy and quick method for extracting IgY from egg yolk.
2. Description of Related Art
When invaded by foreign antigens, the immune systems of vertebrate animals often counteract by synthesis and secretion of antibodies (Abs) interacting with the various epitopes of specific antigens. The collection of these Abs are known as polyclonal antibodies (PAb). Since PAb could bind to their antigens with specificity and high affinity, they are widely applied in basic researches, immunodiagnostic, and immunotherapy.
Traditionally, PAb are produced using bigger mammals, e.g., rabbits and goats, for most of the secreted Abs are distributed in the blood and larger quantities of blood are attainable from these animals. However, there are disadvantages for this traditional way of PAb production, such as the needs of larger space and higher cost for keeping animals and the ineffectiveness in producing Abs against conserved mammalian antigens due to the phylogenetic closeness within mammals. Moreover, because mammalian Abs might interact with human's complement system and rheumatoid factors, the applications of these Abs in serological tests of human samples could encounter unnecessary interference. Furthermore, the repeated and invasive animal bleeding manipulation often incurs ethical concerns from the animal welfare groups.
In response to the challenge of foreign antigens, chickens (Gallus gallus) also produce antibodies. Unlike in mammals, there are only three classes of antibodies identified in chicken, namely IgM, IgA and IgY. Among them, IgY is the predominant form which is continually synthesized, secreted into the blood and transferred to the egg yolk, where it accumulates to a concentration even higher than that in the blood. It has been reported that a single egg contains as much Abs as an average bleed from a rabbit. Since it is cheaper to feed and house chickens than rabbits and a hen can lay eggs often on a daily basis while a rabbit can not be bled with the same frequency, thus, the use of chicken as immunizing hosts is a much more economical and efficient way to produce PAb than the conventional ways. Other than the economical advantage, chickens are not mammals and therefore are more apt to generate Abs against mammalian antigens, including the highly conserved mammalian proteins. Furthermore, IgY will not interact with mammalian's complement system and rheumatoid factors and consequently, the problem of assay interference can be minimized when using IgY in the serological tests of human samples. Other than the advantages described above, PAb production in chicken offers greater compatibility with animal protection regulations for Abs sampling can be easily achieved by egg collection, rather than by the stressful and invasive bleeding manipulation.
Despite PAb production in chicken offers numerous advantages over the conventional ways, it has not been practiced as popular as it should be. One major reason is that egg yolk contains large amount of lipids (ca. 34% by weight) and other irrelevant proteins and this makes IgY extraction difficult and cumbersome. Furthermore, due to the significant difference between their amino acid compositions, the Protein A/G Sepharose columns commonly used for IgG adsorption is inapplicable for IgY purification. Current protocols of IgY extraction from egg yolk often involve a serial precipitation, chromatographic and ultrafiltration steps, which are complicated and time-consuming. Thus, it is desirable to develop a simple and effective method for extracting yolk IgY to good yield and desired purity.